Widely considered the most basal vertebrates alive today, hagfishes represent a pivotal point in evolution and thus offer a key piece to the puzzle of how our lymphocyte-based adaptive immune system evolved. An alternative adaptive immune system has been identified recently in the two extant jawless vertebrates, lampreys and hagfish, which last shared a common ancestor ~480 million years ago. While immunoglobulin- based antigen receptors are used by jawed vertebrates, jawless vertebrates instead use leucine-rich repeat (LRR)-based variable lymphocyte receptors (VLR) of three types to recognize antigens. VLRA and VLRC are assembled in the lamprey thymus-equivalent gill region by two lymphocyte lineages that resemble mammalian ab and gd T cell lineages. VLRB is assembled in hematopoietic tissues by B-like lymphocytes, which undergo plasma cell differentiation and secrete multivalent VLRB antibodies with structural features and antigen specificities that offer significant advantages for biomedical uses. Hagfish cells expressing the different VLRs have not yet been identified or functionally defined. To elucidate the hagfish immune system, monoclonal antibodies specific for invariant portions of their three VLRs have been made for use in analyzing development, distribution and function of hagfish lymphocytes, with particular emphasis on defining the structure and antigen-binding specificities of hagfish VLRB antibodies. The first aim is to define the tissue distribution and gene expression patterns of hagfish VLRA +, VLRB+ and VLRC+ lymphocytes. A principle objective here is to identify the primary sites in which VLRA and VLRB assembly occurs versus secondary sites in which mature lymphocytes undergo proliferation and differentiation. The second aim is to characterize hagfish lymphocyte responses to antigens, allogeneic cells, and mitogens. To determine why hagfish exhibit weak humoral immune responses to immunization, yet have an abundance of circulating VLRB proteins, these experiments will employ refined immunization protocols that elicit robust VLRB antibody responses in lampreys. Pilot studies imply that the hagfish VLRA+ and VLRC+ lymphocytes preferentially respond to cells from other lampreys, and experiments are proposed to find the 'allo-recognition elements' of jawless vertebrates. The third specific aim is to construct yeast surface display libraries for hagfish VLRs for use in large scale surveys of the receptor repertoires and to isolate antigen-specific clones for structural and functional characterization. Comparative screening of the VLRA and VLRB libraries for antigen binding will allow us to test the hypothesis that the VLRA and VLRC repertoires are clonally selected for reactivity with processed antigen. Elucidation of the hagfish immune system will help us to understand how and why lymphocyte development along T- and B- cell pathways occurred during vertebrate evolution and to exploit VLRB antibodies for biomedical purposes.